The kininogens are single-chain glycoproteins which are present in human blood plasma and tissues in two forms: high molecular weight kininogen (120 kDa) and low molecular weight kininogen (64 kDa). A single gene controls the synthesis of both kininogens (Takagaki et al., J. Biol. Chem. 266, 6786 (1985)). The difference between the high molecular weight form and the low molecular weight form is the addition of a unique 56 kDa light chain on high molecular weight kininogen by an alternative mRNA splicing of the single kininogen gene (Kitamura et al., J. Biol. Chem. 260, 8610 (1985); Kakizuka et al., J. Biol. Chem. 265, 10102 (1990)). The presence of the 56 kDa light chain on high molecular weight kininogen gives this form of kininogen unique antigenic and functional properties. The plasma concentration of high molecular weight kininogen is 0.67 micromolar, while the plasma concentration of low molecular weight kininogen is 2.4 micromolar.
There are numerous functions for the plasma kininogens which follow from their structure. The first function of both kininogens is that they serve as the parent proteins for the nonapeptide bradykinin, and for the decapeptide lys-bradykinin. These kinins are the most potent, naturally-occurring vasodilitory mediators. They have profound effects on endothelium, stimulating their prostaglandin synthetic pathways and stimulating release of plasminogen activators. Bradykinin and its derivatives may be major local modulators of the autocrine regulation of blood pressure. Bradykinin is best liberated from high molecular weight kininogen by plasma kallikrein, activated factor XII, factor XIa and plasmin. Low molecular weight kininogen is a better substrate for tissue kallikreins liberating lysbradykinin. Elastase treatment of low molecular weight kininogen makes it a better substrate for kinin release by plasma kallikrein.
Both low and high molecular weight kininogens have identical amino acid sequences from their N-terminus through 12 amino acids beyond the carboxy-terminus of bradykinin. Their so-called "heavy chains" from the amino-terminus of the protein to the amino-terminal end of bradykinin are identical. These heavy chains have been characterized to have three domains (domains 1-3). Domains 2 and 3 contain the amino acid sequence Gln-Val-Val-Ala-Gly (SEQ ID NO:2). This amino acid sequence is highly conserved in evolution in cysteine protease inhibitors (Ohkubo et al., Biochem. 23, 3891 (1984)). Domain 2 uniquely appears to be a good inhibitor of calpains, which are calcium-dependent tissue cysteine proteases (Schmaier et al, J. Clin. Invest. 77, 1565 (1986)). The kininogens' ability to inhibit calpains may have some function in preventing calpain-induced platelet aggregation after thrombin activation (Schmaier et al., Blood 75, 1273 (1990)). Domain 4 on both high and low molecular weight kininogen comprises bradykinin.
High molecular weight kininogen also functions as a cofactor for the activation of the following plasma zymogens: factor XII, prekallikrein, and factor XI. These three plasma zymogens when activated to enzymes, along with high molecular weight kininogen, comprise the proteins of the contact phase of plasma proteolysis. In addition to being a cofactor for activation of each of these plasma zymogens, high molecular weight kininogen is also a substrate of each of their proteolytic forms. The procofactor activity of high molecular weight kininogen is based upon two areas on its unique 56 kDa light chain: First, high molecular weight kininogen has a region on domain 5 which is rich in the basic amino acids glycine, histidine, and lysine that has the ability to bind to anionic surfaces such as kaolin. Secondly, high molecular weight kininogen has a region on its domain 6 which serves as the binding region for prekallikrein and factor XI. Interference with high molecular weight kininogen's ability to bind to negatively charged surfaces with a monoclonal antibody, such as C11C1 (ATCC HB-8964) blocks its procofactor or procoagulant activity (Schmaier et al., J. Biol. Chem. 262, 1405 (1987); U.S. Pat. No. 4,908,431). Similarly, interference with high molecular weight kininogen's ability to bind prekallikrein and/or factor XI by a monoclonal antibody directed to its prekallikrein/factor IX binding region also interfers with its procoagulant activity (Tait et al., J. Biol. Chem. 261, 15396 (1986); Vogel et al., J. Biol. Chem. 265, 12494 (1990)).
It has been a common thought in the contact field that since high molecular weight kininogen has a specific region on its unique light chain that binds to artificial, negatively-charged surfaces, then if this protein interacts with biologic surfaces, e.g., cell membranes, that it does so through the surface binding region contained on domain 5 of its unique 56 kDa light chain. High molecular weight kininogen has been shown to have specific, reversible and saturable binding sites on unstimulated platelets (Gustafson et al., J. Clin. Invest., 78, 310 (1986)), activated platelets (Greengard et al, Biochemistry 23, 6863 (1984)), granulocytes (Gustafson et al., J. Clin. Invest., 84, 28 (1989)), and human umbilical vein endothelial cells (Van Iwaarden et al., 263, 16327 (1988)). The affinity for high molecular weight kininogen to bind to cells in the vascular compartment is between 0.015 and 0.05 micromolar. Since the plasma high molecular weight kininogen concentration is 0.67 micromolar, all intravascular kininogen binding sites should be saturated in vivo. However, the common thought that there could only be a cell-binding region on the light chain of high molecular weight kininogen was shown to be incorrect by the publication that low molecular weight kininogen, the other kininogen which does not contain the 56 kDa light chain that has procoagulant activity, could also specifically, reversibly and saturably bind to human platelets (Meloni et al., J. Biol. Chem. 266, 6786 (1991)). Low molecular weight kininogen's ability to bind to platelets inhibited or was inhibited by high molecular weight kininogen.
Unrelated to the foregoing discussion of the kininogens, treatment of hypertension has consisted of therapy aimed at influencing a number of components involved in blood pressure regulation. .beta.-Adrenergic blockers have been used to decrease hypertension by limiting the extent of cardiac output. .alpha.-Adrenergic antagonists, e.g., .alpha.-methyl dopa, have been utilized to stimulate dilation of arteries. Yet another antihypertensive therapy utilizes nitrate compounds, e.g., nitropaste, to produce venous pooling and arterial dilitation by other means. Finally, inhibitors of kininases, such as captopril, have been used to inhibit one of the seven enzymes that degrade physiologically produced bradykinin. The result is a potentiation of bradykinin's effect by limiting its rate of degradation. None of these standard antihypertensive therapies involves the direct elevation of intravascular bradykinin.
Hereinafter, "human kininogen" shall mean, unless otherwise indicated, both high and low molecular weight forms of any kininogen molecule, in all its various forms derived from human plasma, platelets, endothelial cells, granulocytes, or skin or other tissues or organs, regardless of whether it is found in the fluid or the tissue phase.
"HK" shall mean human high molecular weight kininogen.
"LK" shall mean human low molecular weight kininogen, also known as .alpha.-cysteine protease inhibitor, or .alpha..sup..+-. -thiol protease inhibitor, or .alpha..sub.2 -thiol protease inhibitor.
"Light chain" shall mean, when referring or relating to human kininogen, the 56 kDa intermediate plasma kallikrein-cleavage fragment of HK which has the ability to correct the coagulant defect in total kininogen-deficient plasma.
"Heavy chain" shall mean, when referring or relating to human kininogen, the 64 kDa kallikrein-cleavage fragment of HK or LK, which is free of bradykinin and "light chain".
"D3" or "domain 3" with respect to the kininogen heavy chain shall mean the trypsin-cleavage fragment of the human kininogen heavy chain which is about 21 kDa.
The term "homology" means the degree of identity between two amino acid sequences. For example, 80% homology with respect to a 100-amino acid native polypeptide means that a homologous polypeptide contains identical amino acids when compared to the native polypeptide in any 80 positions out of the 100 amino acid positions of the native polypeptide. By way of further examples, an 80% homologous polypeptide may be generated by any of the following modifications: (i) removing a twenty amino acid sequence from the amino or carboxy terminus of the 100-amino acid native sequence either as a continuous 20-amino acid deletion, or by deleting 20 noncontinuous amino acid residues; (ii) inserting as an internal insertion 20 amino acids into the native 100-amino acid native sequence, either as a continuous 20-amino acid insert, or in isolated inserts comprising one or more amino acids; (iii) adding up to 20 amino acids as an amino- or carboxy terminus of the native sequence; or (iv) any combination of one or more of the aforesaid modifications, the result of which is a homologous sequence of amino acids identical to the native sequence in at least 80 out of 100 positions.